Abstract Glial-derived neurotrophic factor (GDNF) is one of several powerful survival factors for spinal motoneurons that play a key role in sprouting, synaptic plasticity, and reorganization after spinal cord damage. The aim of this study was to investigate the expression of GDNF in plasma of children with spina bifida (SB) and to determine its correlation with both the severity of spinal cord damage and the motor function of these patients. To measure the GDNF expression, we collected plasma samples from 152 children with SB and in 149 matched controls. Endogenous GDNF levels were quantified using a two-site immuno-enzymatic assay. The statistical analysis was performed using the Mann-Whitney two-tailed two-sample test. In children with SB the mean levels of GDNF (131.2 +/- 69.6 pg/mL) were significantly higher (p < 0.001) with respect to the mean levels of the control group (102.7 +/- 6.8 pg/mL). Moreover, in open SB, the GDNF levels (139.2 +/- 81.1 pg/mL) were significantly higher (p < 0.05) with respect to closed SB (117.2 +/- 41.3 pg/mL). In terms of the motor function of patients, we found that in children with poorer motor function, the GDNF levels (134.5 +/- 67.4 pg/mL) were higher, but not statistically significant (p < 0.1), than in patients with better motor outcome (122.3 +/- 72.2 pg/mL). Our study demonstrates GDNF over-expression in children with SB. This upregulation is significantly associated with the severity of spinal cord damage in SB patients and appears to correlate with poor motor function of children, representing an important biochemical marker of the severity of spine injury.